To produce pure enzymes from naturally-occurring sources such as biological liquids, extracts of organs and tissues, cells of microorganisms, fermentation broth, it is necessary to separate the accompanying proteins including other enzymes, nucleic acids and products of decomposition thereof, low-molecular compounds and salts. Conventional methods of recovery and purification of enzymes are based on differences in physicochemical properties of enzymes and other components of the mixture to be separated such as molecular weight, charge, and solubility. These methods are exemplified by fractional precipitation of proteins by means of salts and organic solvents; gel-filtration, ultra-filtration, on-exchange chromatography. However, each of the prior art methods is insufficiently effective. Satisfactory results are obtained by combination of several methods which, however, is associated with great losses of enzymes and increased production and labor costs. The method of biospecific or affinity chromatography principally differs from the traditional methods in that it is based on the use of the most individualized property of an enzyme, i.e. its ability of selectively reacting with substrates or inhibitors resembling the substrates in the structure thereof.
Addition of a ligand, i.e. substrate or an analogue thereof to an inert carrier provides a specific sorbent which is capable of selectively combining, from a complex mixture of compounds, an enzyme only. After washing the sorbent with a suitable solution for elimination of impurities, the enzyme retained by the sorbent is desorbed by means of a solution which is selected so as to dissociate the enzyme-ligand complex (variation of pH, elevation of the salt concentration, addition of organic solvents). The resulting enzyme product is generally enriched, to a substantial degree, with the desired enzyme and frequently it comprises a pure enzyme. The method makes it possible to substantially increase yields of enzymes and considerably improve quality of purification thereof.
Also known in the art are methods for purification and isolation of proteolytic enzymes which are based on the lon-exchange chromatography. Special mention should be made of the method based on chromatography on modified silochromes. However, this method is insufficiently selective, and is unsuitable for certain technical applications such as recovery of enzymes at a high yield directly from a fermentation broth or gastric juice, i.e. from mixtures with a low concentration of an active enzyme containing a large amount of inorganic and organic impurities including colored ones.
Further known in the art is a method for recovery and purification of neutral and alkaline proteinases from a brewing wort by means of a phenol-formaldehyde sulphocationite Duolite 10. The purification process involves 6 successive stages in addition to dialysis and lyophilization, i.e. double precipitation of the protein fraction with acetone, followed by centrifugation, double chromatography on a DEAE-cellulose, purification on Duolite 10 and precipitation by means of ammonium sulphate.
Duolite 10 in this case is used for the removal of colored impurities from the mixture, not for a direct separation of individual enzymes. The final object of using it is separation of a mixture of neutral and alkaline proteinases with a yield ranging from 33 to 67%.
In this method for recovering enzymes, biospecific sorbents are most extensively used which are prepared from agarose activated by bromocyane and covalently bonded with compounds specific for this particular class of enzymes, i.e. ligands.
In the prior art methods for purification of enzymes use is made of biospecific sorbents prepared by interaction of Sepharose 4B activated with cyanogen bromide with such peptide ligands as methyl ether and amide of .epsilon.-aminocapronyl-D-phenylalanine, methyl ether of .epsilon.-aminocapronyl-L-phenylalanine-D-phenylalanine; N-2,4-dinitrophenylhexamethylene diamine and an antibiotic, i.e. Gramicidin S. This method, however, has certain disadvantages. The main disadvantage resides in that the above-mentioned ligands (except Gramicidin S) can be obtained only by an extensive multi-stages synthesis, whereby the preparation of sorbents is hampered.
Also known in the art is a method for purification of enzymes with the use, as biospecific sorbents, of Sepharose 4B covalently bonded with an antibiotic Gramicidin S (cf. "Biokhimija" (Biochemistry), vol. 41, 2, 294; 1976).
A sorbent containing, as a ligand, a known antibiotic Gramicidin S is more readily available, since the latter is prepared in a relatively simple manner. At present, Gramicidin C, however, finds limited application in medicine and is produced in small quantities. Disadvantages of a sorbent based on Gramicidin S-Sepharose 4B may be exemplified by difficulties encountered in the synthesis of the sorbent owing to a very low solubility of Gramicidin S. Reaction of Gramicidin S with Sepharose 4B activated with bromocyane is conducted in solutions with a high content of dimethylformamide which might partly destroy the structure of Sepharose. In the sorbent synthesis Gramicidin S settles, thus reducing the final product yield and hindering the product purification. Furthermore, the range of application of Gramicidin S-Sepharose 4B is limited to certain classes of proteinases and does not involve practically important serine proteinases.
It is an object of the present invention to overcome the above-mentioned disadvantages.
It is an object of the present invention to provide such a method which makes it possible to selectively recover and purify proteolytic enzymes from naturally-occurring sources.
It is another object of the present invention to provide a simplified method for purification of proteolytic enzymes.